Numerous methods have been reported for detecting nucleic acids having a specific base sequence that consist of investigating the base sequence of a nucleic acid using artificially synthesized short-stranded oligonucleotides such as probes or primers. In particular, various techniques using fluorescent light have been developed based on its superior detection sensitivity in genetic analyses such as analyses of somatic mutations and single nucleotide polymorphisms.
One example of a method for identifying nucleic acids using an FRET probe is a so-called molecular beacon method that uses a single-stranded nucleic acid having mutually complementary base sequences on the 5′-terminal side and 3′-terminal side and in which both ends are labeled with a fluorescent substance and a quenching substance, respectively (molecular beacon probe) (see, for example, Japanese Unexamined Patent Application, First Publication No. 2005-098876). Although a quenched state results due to conjugation of both ends resulting in the formation of an intramolecular loop in the case only the molecular beacon is present; as a result of hybridizing with a target nucleic acid, the intramolecular loop is unlocked resulting in the emission of fluorescent light. Genetic polymorphisms can be identified by using a molecular beacon that specifically hybridizes with a specific genotype among genetic polymorphisms (see, for example, Japanese Unexamined Patent Application, First Publication No. 2008-292371).
In addition, there are also methods that detect nucleic acids using a FRET probe obtained from a fluorescent intercalator and fluorescent probe (see, for example, Japanese Patent No. 4023523). In these methods, a fluorescent probe labeled with a fluorescent substance hybridizes with another single-stranded nucleic acid, and a fluorescent intercalator is inserted between the base pairs of the double-stranded nucleic acid formed. By using FRET that occurs between the fluorescent intercalator and the fluorescent substance used to label the fluorescent probe, a distinction can be made between the fluorescent probe when present alone and the fluorescent probe that forms a double-stranded nucleic acid. By using this principle, genetic polymorphisms can be identified by labeling a probe that specifically hybridizes with a specific genotype among genetic polymorphisms and a probe that specifically hybridizes with another genotype with respectively different fluorescent substances, and then detecting FRET occurring between each of the fluorescent substances and a fluorescent intercalator (see, for example, International Patent Publication No. WO 2008/080417).
On the other hand, due to the advancement of photometric technology in recent years, it has become possible to detect and measure faint light at the level of a single photon or single fluorescent molecule using confocal microscope optics and ultra-high sensitivity photodetection technologies capable of photon counting (detection of individual photons). Various devices or methods have been proposed for detecting molecular interaction, or bonding and dissociation reactions of biomolecules using these technologies for measuring faint light. For example, in the case of fluorescent correlation spectroscopy (FCS: see, for example, Japanese Unexamined Patent Application, First Publication No. 2005-098876; Japanese Unexamined Patent Application, First Publication No. 2008-292371; Japanese Unexamined Patent Application, First Publication No. 2007-20565; Japanese Unexamined Patent Application, First Publication No. 2008-116440; and Japanese Unexamined Patent Application, First Publication No. H4-337446), laser confocal microscope optics and photon counting technology are used to measure fluorescence intensity from fluorescent molecules or fluorescence-labeled molecules that enter and leave a microregion in a sample solution (focal region where laser light from a microscope is concentrated, also referred to as confocal volume). Information such as the speed of movement, size or concentration of fluorescent molecules and the like can be acquired, or various phenomena can be detected, including changes in molecular structure or size, molecular bonding and dissociation reactions, dispersion and aggregation, based on the average retention time (translational diffusion time) of fluorescent molecules and the average value of the number of molecules remaining in the microregion determined from the value of the autocorrelation function of the measured fluorescence intensity. In addition, in the case of fluorescence intensity distribution analyses (FIDA: see, for example, Japanese Patent No. 4023523) and photon counting histograms (PCH: see, for example, International Patent Publication No. WO 2008/080417), a histogram is generated of the fluorescence intensity of fluorescent molecules and the like that enter and leave a confocal volume measured in the same manner as FCS, and the average value of brightness characteristic to those fluorescent molecules and the average value of the number of molecules remaining in the confocal volume are calculated by fitting a statistical model to the distribution of that histogram. Changes in molecular structure or size, bonding and dissociated states, dispersion, aggregation and the like are then estimated based on this information. Moreover, Japanese Unexamined Patent Application, First Publication No. 2007-20565 and Japanese Unexamined Patent Application, First Publication No. 2008-116440 propose a method for detecting a fluorescent substance based on the elapsed time of a fluorescent signal of a sample solution measured using confocal microscope optics. Japanese Unexamined Patent Application, First Publication No. H4-337446 proposes a signal arithmetic processing technology for detecting the presence of fluorescent microparticles in a flow or on a substrate by using photon counting technology to measure faint light from fluorescent microparticles that have passed by a flow cytometer or from fluorescent microparticles immobilized on the substrate.
In particular, according to methods employing technologies for measuring fluorescence in a microregion using confocal microscope optics and photon counting technology in the manner of FCS or FIDA and the like, the sample required for measurement is of a much lower concentration and extremely small amount in comparison with in the past (the amount used for a single measurement is at most about several tens of microliters), and measurement time is shortened considerably (measurements taking only several seconds per measurement can be repeated several times). Thus, these technologies are expected to be powerful tools for enabling experimentation or testing to be carried out inexpensively or rapidly in comparison with conventional biochemical methods particularly in the case of carrying out analyses on scarce or expensive samples frequently used in medical, biological and other research and development fields, or in the case of large numbers of specimens as in the clinical diagnosis of diseases or screening for physiologically active substances.